ABSTRACT Carpal tunnel syndrome (CTS) is the most common hand disorder, and nearly half a million carpal tunnel release surgeries are performed annually in the United States. During surgery, the transverse carpal ligament (TCL) is transected to decompress the median nerve. However, transecting the TCL reduces grip strength, causes pillar pain, results in greater carpal bone motion, and may damage the surrounding nerves, vessels or tendons. To avoid these surgical complications, innovative treatment solutions with non-operative approaches are sorely needed. Transection of the TCL as a surgical treatment for carpal tunnel syndrome is analogous to surgical fasciectomy for Dupuytren's contracture. As collagenase injection successfully treats Dupuytren's contracture, it is compelling to explore this collagenolytic effect on the TCL. Our long-term goal is to develop a novel non-operative treatment for carpal tunnel syndrome by enzymatically degrading the TCL. The objective of this project is to inject collagenase into the TCL and evaluate its enzymatic effect on TCL morphology and mechanics using state-of-the-art robotic and ultrasound technologies. Our central hypothesis is that collagenase effectively degrades the TCL, leading to decreased TCL thickness and stiffness in vitro as well as increased TCL length and arch area in situ. This hypothesis will be tested with two specific aims: (1) to investigate dose- and time-dependent collagenolytic effects on the morphological and mechanical properties of the TCL in vitro, and to identify an optimal collagenase dose that can achieve effective and safe collagenolysis of the TCL after 24 hours; (2) to examine the changes in structural properties of the TCL in situ in response to collagenase injections and determine an injection configuration to achieve TCL elongation of 2 mm. In Aim 1, collagenase at various doses will be injected into TCL tissues dissected from cadaveric hands. B-mode and acoustic radiation force impulse (ARFI) ultrasound imaging will be performed to measure thickness and stiffness changes over time up to 24 hours. The stiffness of the TCL will be derived from shear wave velocity measured by ARFI. We will also identify the minimum effective collagenase dose that can achieve 80% reduction of thickness and shear wave velocity at 24 hours. In Aim 2, collagenase will be injected using the optimal dose determined in Aim 1 along the longitudinal midline of the TCL in situ. Carpal tunnel pressure will be applied to obtain local strain at the injection sites and gross tissue elongation, which will be used to determine an injection to achieve the desired TCL elongation. Robot-assisted collagenase injection and ultrasound scanning will be performed to achieve precise injection and reproducible scanning for TCL reconstruction. The implementation of the proposed project is the critical first step to exploring the possibility of collagenolysis of the TCL as a novel non-operative treatment for CTS. The knowledge obtained from this project will guide future in situ carpal tunnel studies, in vivo animal studies, and clinical trials, tapping into the potential of collagenase injection as a novel non-operative treatment for CTS.